The present invention relates to the discovery of the functional receptor (TWEAKR) for the TWEAK protein. More particularly, the invention relates to the use of TWEAKR agonists and antagonists in methods of treatment, and to screening methods based on TWEAKR and the TWEAK-TWEAKR interaction.
A. Angiogenesis
Angiogenesis is a multi-step developmental process that results in the formation of new blood vessels off of existing vessels. This spatially and temporally regulated process involves loosening of matrix contacts and support cell interactions in the existing vessels by proteases, followed by coordinated movement, morphological alteration, and proliferation of the smooth muscle and endothelial cells of the existing vessel. The nascent cells then extend into the target tissue followed by cell-cell interactions in which the endothelial cells form tubes which the smooth muscle cells surround. In a coordinated fashion, extracellular matrix proteins of the vessel are secreted and peri-endothelial support cells are recruited to support and maintain structural integrity (see, e.g., Daniel et al., Ann. Rev. Physiol. 2000(62):649, 2000). Angiogenesis plays important roles in both normal and pathological physiology.
Under normal physiological conditions, angiogenesis is involved in fetal and embryonic development, wound healing, organ regeneration, and female reproductive remodeling processes including formation of the endometrium, corpus luteum, and placenta. Angiogenesis is stringently regulated under normal conditions, especially in adult animals, and perturbation of the regulatory controls can lead to pathological angiogenesis.
Pathological angiogenesis has been implicated in the manifestation and/or progression of inflammatory diseases, certain eye disorders, and cancer. In particular, several lines of evidence support the concept that angiogenesis is essential for the growth and persistence of solid tumors and their metastases (see, e.g., Folkman, N. Engl. J. Med. 285:1182, 1971; Folkman et al., Nature 339:58, 1989; Kim et al., Nature 362:841, 1993; Hori et al., Cancer Res., 51:6180, 1991). Angiogenesis inhibitors are therefore useful for the prevention (e.g., treatment of premalignant conditions), intervention (e.g., treatment of small tumors), and regression (e.g., treatment of large tumors) of cancers (see, e.g., Bergers et al., Science 284:808, 1999).
There is a need for additional compositions and methods of modulating angiogenesis for the prevention, abrogation, and mitigation of disease.
B. TWEAK
The TWEAK protein, which has also been called TREPA and Apo3L, is a member of the tumor necrosis factor (TNF) family and is expressed in a wide variety of human tissues (Chicheportiche et al., J. Biol. Chem., 272(51):32401, 1997; see also Wiley, PCT Publication No. WO 98/35061, 13 Aug. 1998). Like most TNF family members, TWEAK is a Type II membrane protein with an extracellular C-terminal domain. Although TWEAK was originally described as a weak inducer of apoptosis, this induction of cell death was later shown to be indirect (Schneider et al., Eur. J. Immunol. 29:1785, 1999).
Lynch et al. demonstrated that TWEAK directly induces endothelial cell proliferation and angiogenesis (J. Biol. Chem., 274(13):8455, 1999). Picomolar concentrations of recombinant soluble TWEAK induce proliferation in multiple endothelial cell lines and in aortic smooth muscle cells, and reduce the requirement for serum and growth factors in culture. Moreover, TWEAK induces a strong angiogenic response in a rat corneal pocket assay. Since TNF family members initiate biological responses by signaling through members of the TNF receptor family, there has been great interest in identifying and characterizing the TWEAK receptor.
Marsters et al. reported that TWEAK binds to and signals through a death-domain containing receptor known variously as DR3, Apo3, WSL-1, TRAMP, or LARD (Marsters et al., Current Biology 8(9):525, 1998). Schneider et al., however, showed that TWEAK binds to and signals in Kym-1 cells but that Kym-1 cells do not express the receptor DR3 (Schneider et al., Eur. J. Immunol. 29:1785, 1999). These results suggest the existence of a yet to be identified TWEAK receptor.
Because TWEAK induces angiogenesis in vivo, there is a particular need to identify the major functional TWEAK receptor. Once identified, the TWEAK receptor may be used to screen for and develop TWEAK receptor agonists and antagonists for the modulation of angiogenesis and the treatment of human disease.
The present invention is based upon the identification and biological characterization of the major functional TWEAK receptor. As described below, cDNA encoding the TWEAK receptor was molecularly cloned from a human endothelial cell expression library.
Although DNA and deduced amino acid sequences corresponding to the TWEAK receptor identified herein have been reported (see, e.g., Kato et al., PCT Publication No. WO 98/55508, 10 Dec. 1998 and Incyte, PCT Publication No. WO 99/61471, 2 Dec. 1999), it was not heretofore appreciated that these sequences encode a receptor for TWEAK or that the encoded polypeptide is involved in modulating angiogenesis. Similarly, investigators have recently claimed methods of making and using TWEAK receptor antagonists to treat immunological disorders, but without identifying the major TWEAK receptor or its role in angiogenesis (Rennert, PCT Publication No. WO 00/42073, 20 Jul. 2000). These deficiencies have been addressed, as described herein, by identification of the major TWEAK receptor (TWEAKR) and characterization of its biological activities. The identification of TWEAKR has led to the development of compositions for the modulation of angiogenesis, and also provides screening tools for the identification of diagnostics and therapeutics.
The invention provides methods of modulating angiogenesis in a mammal in need of such treatment comprising administering a therapeutically-effective amount of a composition comprising a TWEAK receptor antagonist or TWEAK receptor agonist. The composition preferably comprises a pharmaceutically acceptable carrier and the mammal is preferably a human.
In some more preferred embodiments the composition inhibits angiogenesis and comprises a TWEAK receptor antagonist, such as a soluble TWEAK receptor fragment, an antagonistic antibody, or an antagonist that disrupts the interaction between the TWEAK receptor and a TRAF molecule. In some most preferred embodiments the antagonist comprises amino acids 28-79 of SEQ ID NO:7 or amino acids 28-309 of SEQ ID NO:7. The TWEAK receptor antagonists are preferably used to treat a mammal that has a disease or condition mediated by angiogenesis, more preferably a disease or condition characterized by ocular neovascularization or a solid tumor. In some embodiments, the mammal is further treated with radiation or with a second chemotherapeutic agent.
In some more preferred embodiments the composition promotes angiogenesis and comprises a TWEAK receptor agonist, such as an agonistic antibody. The TWEAK receptor agonists are preferably used to treat a vascularization deficiency in cardiac or peripheral tissue, to enhance wound healing or organ transplantation, or in conjunction with bypass surgery or angioplasty.
The invention also provides antagonists comprising a soluble TWEAK receptor fragment for use in medicine, preferably comprising amino acids 28-79 of SEQ ID NO:7 or amino acids 28-309 of SEQ ID NO:7, as well as nucleic acids encoding soluble TWEAK receptor fragments. And the invention provides for the use of a composition comprising a TWEAK receptor antagonist or TWEAK receptor agonist for the preparation of a medicament for modulating angiogenesis in a mammal in need of such treatment.
The invention further provides methods of identifying a compound that is capable of modulating angiogenesis, including: (a) identifying a test compound that binds to a TWEAK receptor extracellular domain, wherein the test compound is not TWEAK; (b) identifying a test compound that affects the interaction between a TWEAK and a TWEAK receptor; and (c) identifying a test compound that modulates the interaction between a TWEAK receptor and a TRAF. The invention encompasses compounds identified according to these methods.
The invention also provides a method for targeting a detectable label or chemotherapeutic to vascular tissue comprising contacting the vascular tissue with an antibody that binds TWEAK receptor. In some preferred embodiments the antibody is conjugated to a radioisotope, chemiluminescent or fluorescent compound, or enzyme. In some preferred embodiments the antibody is conjugated to a cytotoxin.